1. Field of the Invention
The present invention provides a three-dimensional image obtaining device and a method thereof for visualizing, as a specimen of a biological material having low light absorption and being almost transparent (hereinafter referred to as a biological specimen), a biological specimen of a cell and a microorganism, a sliced organ/tissue specimen.
The invention particularly relates to a three-dimensional image obtaining device applicable as an observation module forming a part of a microscope apparatus and a processing apparatus or a biomedical related measurement apparatus, and to a method thereof.
2. Description of the Related Art
There are demands for obtaining a three-dimensional image of a biological specimen such as a cell without dyeing using a florescent dye or the like. Specifically, there are demands that the observer can actually see not only the outer shape of a cell but also organelles such as nucleus in a cell.
Since the aforementioned biological specimen has low light absorption and scattering ratio and is almost transparent, it shows almost no contrast in a microscope for observing with a transmitted light. Accordingly, a phase contrast microscope or a differential interference contrast microscope which gives contrast to a biological specimen is used for observing such a specimen. An image of the biological specimen observed with these microscopes is a two-dimensional projected image and not a three-dimensional image.
Document 1 (Holmes T J, O'Connor N J, Blind deconvolution of 3D transmitted light brightfield micrographs, Journal of Microscopy, 2000, vol. 200, pt. 2, p. 114-127) discloses a microscope apparatus for detecting transmitted light intensity using an optical system of a confocal microscope. This apparatus makes a three-dimensional transmitted light map of a biological specimen, and makes a three-dimensional image of the specimen by a blind deconvolution. However, the biological specimen is almost transparent and absorbs a minute amount of light. Therefore, the three-dimensional transmitted light map only has a slight change from a three-dimensional transmitted light map when there is no specimen, and it is difficult to measure with high S/N. There are demanded an optical system that is most suitable for measuring a specimen that is almost transparent and a novel device for detecting a minute light attenuation amount.
Computed tomography (CT) using a cone beam has been reported as another method to obtain a three-dimensional image from a transmitted light. However, the CT method requires providing a rotating mechanism for shooting plural times while changing the shooting angle.
In recent years, a confocal laser scanning microscopes has been developed. JP H7-199073A discloses a confocal laser scanning microscope and an optical tomographic image displaying method thereof. The confocal laser scanning microscope can obtain a three-dimensional image when used in combination with a fluorescent dye. However, it cannot obtain a three-dimensional image in a non-dyed state.
Further, JP H9-179037A discloses a method of imaging a specific substance with a transmission laser microscope. This method is capable of obtaining a three-dimensional image. However, even with this microscope, it is not possible to image a biological specimen that is almost transparent without dyeing.
Further, JP H9-304178A discloses a transmittancy measurement technique aiming at highly accurate measurement that is not affected by inputted light intensity. The transmittancy measurement technique weights electrical signals of two light receivers and subtracts them, so as to obtain a transmittancy measurement output that becomes zero. This technique potentially has a possibility of measuring an average transmittancy of an entire specimen that is almost transparent. The disclosed technique passes a parallel optical beam through a substance to which a light is transmitted, so as to obtain an average value of the entire transmitted region. Even if a scanning function is added, the obtained image is a projected image, and a three-dimensional image is not obtained.
Furthermore, JP H9-297269A discloses a two-dimensional scanning type image input apparatus and a scanning probe microscope with black level correction. However, a specimen that is almost transparent cannot be observed by transmission.
A main object of the present invention is to provide a three-dimensional image obtaining device and a method thereof, in which a biological material that is almost transparent such as a cell is mounted in a non-dyed state as a specimen, and which are most suitable for an observation apparatus such as a microscope for visualizing a low-contrast specimen.
Another object of the present invention is to provide an apparatus for performing observation of variation over time, optical stimulation, and optical processing of a three-dimensional arbitrary point in a specimen observation region of a biological specimen that is almost transparent, which is visualized using the above three-dimensional image obtaining device.